Mechanism Of Cadmium Induced Toxicity In Testis

The rodent testes are generally more susceptible to cadmium(Cd)-induced toxicity than the other tissues, however the molecular mechanism is not unclear up-to-data. For a long time, it has been considered that impaired germinal epithelium induced by acute lack of blood was the primary cause in testes of rat treated with cadmium. However, a low dose of cadmium actually did not result in visible vascular lesions in the testes, but resulted in failure of spermiation in the seminiferous epithelium. These facts suggest that there are other causes in the development of Cd-induced toxicity in testes. Several authors reported that the teste had shown either no induction or a reduced expression of the metallothionein (MT) gene when animals were exposed to Cd. It has also been reported that testicular Cd is bound to a protein different from MT. The testicular Cd-binding protein contains less cysteine and more glutamate than MT. However, some other studies have shown that MT is constitutively expressed in the whole testes or specific testicular cells at levels higher than in some other organs, e.g. liver, and that in vivo or in vitro Cd exposure increases MT gene expression in the testes. Therefore, the published data are inconclusive as to whether MT exists in the testes under physiological conditions. We now clearly demonstrated by RT-PCR analysis and DNA sequencing of the cloned PCR products that the MT1 and MT2 genes are transcribed as their respective mRNA in the rat testis.The results of Iida, et al. have shown that MT gene expression appears to be not only tissue specific but also cell specific. However, most of the studies on MT gene expression and synthesis have been focused on the whole testes or only one type of the cells, which may mask the results on MT activity in individual cell types, since they contribute to various ratio of parenchymal volume (interstitial cell: 11%, sertoli cells 2~3%, and spermatogenic cell 80%)On the other hand, MT gene expression is time-dependent. It was reported that the levels of both MT mRNA isoforms in rat liver were substantially increased 4~6 h after Cd treatment, followed by a reduction and chromatography demonstrated asignificant time-related increase in MT protein level. In vitro studies show that MT induction is in a dose- and time-dependent manner in both interstitial and sertoli cells. Some of the controversy may derive from the different study time.In general, the studies on Cd-induced MT gene expression and MT synthesis in the testes, have been carried out under acute exposure to toxic Cd levels. It is also conceivable that the biochemical changes occurring in the testes under such conditions of Cd toxicity might mask the molecular processes of MT gene expression under a lower Cd exposure dose.Our previous studies have demonstrated that the inability to induce the metal-detoxicating MT in response to Cd might account for the higher susceptibility of testes to Cd toxicity relative to liver. We also found that increased MT mRNA was not translated into corresponding protein, and the reason is unclear. One hypothesis is that the other genes and proteins are probably involved in the absence of an increase in the MT gene translation product in the testes of Cd-treated animals. In order to clarify the molecular mechanisms of Cd-induced toxicity and to further understand the spermatogenesis, systemic study on Cd-induced genes expression may be one important approach.The present study examined whether MT exists in the testes and whether MT gene expression is tissue specific or cell specific or time dependent. In addition, identification and analysis of differential gene with Affymetrix rat U34 arrays and protein expression with two-dimensional electrophoresis may helpful systematically to explain the related mechanisms involved in the Cd-induced testicular toxicity. 1. Expression of metallothionein mRNA in testes of rat treated with low cadmiumMTl and MT2 mRNA levels were determined by reverse transcriptional PCR followed by densitometry scanning, and the DNA sequences were examined. The data clearly indicate that the MTl and MT2 genes are constitutively expressed in the rat testis. However, there were no difference in transcription of MTl and MT2 between testes of rats treated with low cadmium and the controls (8 day, e.o.d ). This indicated that expression of MT mRNA may be time dependent.2. Metallothionein gene expression under different time in testis and liver of rats treated with cadmiumIn order to clarify whether the MT expression is time dependent, Cd-induced MT gene expression and MT protein accumulation under different time in testes and liver were compared. MT1 and MT2 mRNA levels were determined by reverse transcriptional PCR followed by densitometry scanning, and MT was quantitated by the ELISA method. Testes had higher levels of MT1 and MT2 mRNAs in 1 h, 3 h, 6 h and 24 h over control, but MT protein did not increase. Cd exposure increased hepatic MTs mRNA and MT protein. Thus, These results indicate that, although Cd exposure results in increases of MT mRNA in testes. it does not enhance MT synthesis. The inability to induce the metal-detoxicating MT-protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative toliver.3. Cell- and time-dependent metallothionein gene expression in testes of rats treated with cadmiumIn order to clarify whether the MT expression is cell specific, we compared metallothionein (MT) gene expression, MT protein accumulation, and Cd retention under different time in freshly isolated single testicular cell types (sertoli, interstitial and spermatogenic cells) and liver of rats treated with Cd. In the present study, we demonstrated that the induction of MT1 mRNA was lower than MT2 mRNA in sertoli cells and liver of rat treated with Cd, but it was opposite in interstitial cells. Furthermore, the MT1 mRNA levels of spermatogenic cells decreased 0-3 h after Cd treatment, followed by an increase; in contrast, MT2 mRNA levels increased 0-3 h after Cd treatment, followed by a reduction, but induced-extents of them were lower than those of others. Cd exposure substantially increased hepatic MT, but did not increase MT translation in testicular three cell types. These results indicate: (1) that Cd-induced metallothionein mRNA expression is cell- and time-dependent; (2) that the inability to induce the metal-detoxicating MT-protein in response to Cd, might account for higher susceptibility of testes to Cd toxicity and carcinogenesis relative to liver.4. cDNA arrays associated with low cadmium toxicity in testes of ratscDNA arrays and real-time quantitative polymerase chain reaction (real-time PCR) technique were used to clarify the cause of the inability to induce the metal-detoxicating MT-protein in response to Cd. RESULTS: Newly identified genes that were regulated in expression level between testes of rats treated with cadmium (4umol Cd/kg) and control implicated in cell energy metabolism, defense responses and DNA repair. Our studies for the first time demonstrated that expression of T-kininogen, calmegin, UDP-glucuronosyl transferase, heme oxygenase and mismatch repair protein gene were associated with cadmium toxicity. In addition, vitamin C at the concentration of 400 mg/kg obviously attenuated cadmium-induced toxicity in testes of rats. Conclusion: Cd-induced toxicity and carcinogenesis probably acts through energy metabolisms, defense responses, gene regulation and DNA repair and vitamin C may play essential roles in protecting testes of rats exposed to cadmium.5. Proteomic profile associated with low cadmium toxicity in testes of ratsCd is an environmental and industrial pollutant that affects the male reproductive system of human and animals. To identify proteins involved in cadmium induced testicular toxicity, we used two-dimensional electrophoresis and MALDI-TOF-MS technique. Newly identified proteins that were regulated in expression level between testes of rats treated with cadmium (4 umol Cd/kg) and control implicated in cell energy metabolisms, defence responses, gene regulation and DNA repair. Our studies for the first time demonstrated that expression of carnitine, O-palmitoyltransferase II, voltage-dependent anion channel 2, dihydrolipoamide dehydrogenase, 3-phosphoglycerate dehydrogenase, serine protease inhibitor 3 and LIM-only protein ACT were associated with cadmium toxicity. Interestingly, many of them are related to energy metabolisms and are down regulated. However, as the organelles that produce energy, mitochondria are not affected in respiratory chain at low cadmium treatment. In addition, vitamin C at the concentration of 400 mg/kg obviously attenuated cadmium-induced toxicity in testes of rats, indicating that vitamin C may...